KIT ATP-Binding Pocket/Activation Loop Mutations in GI Stromal Tumor: Emerging Mechanisms of Kinase Inhibitor Escape

Abstract

Purpose: Imatinib resistance in GI stromal tumors (GISTs) is primarily caused by secondary KIT mutations, and clonal heterogeneity of these secondary mutations represents a major treatment obstacle. KIT inhibitors used after imatinib have clinical activity, albeit with limited benefit. Ripretinib is a potent inhibitor of secondary KIT mutations in the activation loop (AL). However, clinical benefit in fourth line remains limited and the molecular mechanisms of ripretinib resistance are largely unknown.

Patients and methods: Progressing lesions of 25 patients with GISTs refractory to ripretinib were sequenced for KIT resistance mutations. Resistant genotypes were validated and characterized using novel cell line models and in silico modeling.

Results: GISTs progressing on ripretinib were enriched for secondary mutations in the ATP-binding pocket (AP), which frequently occur in cis with preexisting AL mutations, resulting in highly resistant AP/AL genotypes. AP/AL mutations were rarely observed in a cohort of progressing GIST samples from the preripretinib era but represented 50% of secondary KIT mutations in patients with tumors resistant to ripretinib. In GIST cell lines harboring secondary KIT AL mutations, the sole genomic escape mechanisms during ripretinib drug selection were AP/AL mutations. Ripretinib and sunitinib synergize against mixed clones with secondary AP or AL mutants but do not suppress clones with AP/AL genotypes.

Conclusion: Our findings underscore that KIT remains the central oncogenic driver even in late lines of GIST therapy. KIT-inhibitor combinations may suppress resistance because of secondary KIT mutations. However, the emergence of KIT AP/AL mutations after ripretinib treatment calls for new strategies in the development of next-generation KIT inhibitors.

FIG 1.

Summary of KIT mutations in progressing lesions of KIT-mutant GISTs. (A) Oncoprint for sequencing results of GIST biopsies in 25 consecutive patients with progression on ripretinib. Bold type signifies primary mutations. For AP and AL mutations, amino acid substitutions are shown in each column. Patients are sorted by time to progression on ripretinib. Ripretinib dose is displayed as standard 150 mg once daily (S) and escalated (100mg or 150mg twice daily [E]). The last row shows the number of treatment lines, including ripretinib, before KIT sequencing. (B-C) Frequency of AP, AL, and AP/AL mutations in patients with two or more KIT mutations in (B) a historical preripretinib cohort from three institutional databases in comparison with (C) the postripretinib progression cohort described in A. Bold type = primary mutation; continuous line = in-cis mutation by cDNA long-read sequencing; dotted line = allelic frequency of AP and AL mutations consistent with in-cis. ^aTwo clones of the same metastasis. AL, activation loop; AP, ATP-binding pocket; delins, see Appendix Table A1. ECD, extracellular domain; GIST, GI stromal tumor; JMD, juxtamembrane domane; TTP, time to progression; Tx lines, number of treatment lines before sequencing.

FIG 2.

Novel GIST cell line models recapitulate clinical ripretinib liabilities and display reciprocal sensitivity to sunitinib. (A and B) Summary of (A) ripretinib and (B) sunitinib GR50 values (nM) at 72 hours for our representative panel of GIST cell lines. The x-axis denotes the location of the primary mutation; green font signifies primary mutations only. Colored ellipses signify locations of respective mutations. (C) Immunoblots of KIT and KIT phosphorylation after ripretinib treatment in relevant isogenic cell lines. DMSO, dimethyl sulfoxide; GIST, GI stromal tumor.

FIG 3.

Drug screens indicate therapeutic vulnerabilities of AP/AL-mutated KIT. (A) Compound screen with 55 FDA-approved TKIs at 100 nM and 72 hours of treatment in different isogenic GIST cell lines. Cell growth was normalized to DMSO-treated cells at 0 and 72 hours for each cell line. AL, activation loop; AP, ATP-binding pocket; DMSO, dimethyl sulfoxide; FDA, US Food and Drug Administration; GIST, GI stromal tumor; TKI, tyrosine kinase inhibitor.

FIG 4.

Evaluation of kinase inhibitors approved for GIST treatment in addition to NIN, Bezu, IDRX-42 (IDRX), and NB003 in KIT- and PDGFRA-driven GIST cell lines. Nanomolar GR50 values after 72 hours of treatment are depicted. Mutation denotes the mutated protein domain responsible for sensitivity or resistance. AL, activation loop; AP, ATP-binding pocket; Ava, avapritinib; Bezu, bezuclastinib; ECD, extracellular domain; GIST, GI stromal tumor; IM, imatinib; JMD, juxtamembrane domane; NIN, nintedanib; RE, regorafenib; Rip, ripretinib; SU, sunitinib.

FIG 5.

Impact of combined treatment with AP and AL inhibitors. Different cell lines and a balanced mixed culture were treated with sunitinib 100 nM, ripretinib 100 nM, or a combination of both drugs (both 100 nM) for 72 hours. Cell growth was normalized to 0 hours and 72 hours of DMSO treatment. AL, activation loop; AP, ATP-binding pocket; DMSO, dimethyl sulfoxide.

FIG 6.

Different clinical scenarios and clonal outcomes after progression on ripretinib. Patients with disease that is dominated by AL mutations—typically selected for after sunitinib treatment—will likely develop AP/AL mutations. In this study, these mutations were particularly common in patients with prolonged clinical benefit from ripretinib, indicating this clinical scenario. Given the lack of any known treatment against these novel compound mutations, local therapies may help to contain these clones, but the clinical benefit of a local treatment needs to be confirmed. Patients with mixed clones will have AP-mutant clones escape on ripretinib, depending on the extent of affected metastases. Treatment with ripretinib or sunitinib could still benefit patients, depending on the dominance of progression—as would a combination (experimental only) or metronomic treatment. AL, activation loop; AP, ATP-binding pocket; PD, progressive disease; Tx, treatment.